The following description of the background of the invention is provided to aid in understanding the invention, but is not admitted to describe or constitute prior art to the invention.
Cellular signal transduction is a fundamental mechanism whereby extracellular stimuli are relayed to the interior of cells and subsequently regulate diverse cellular at processes. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins. Phosphorylation of polypeptides regulates the activity of mature proteins by altering their structure and function. Phosphate most often resides on the hydroxyl moiety (xe2x80x94OH) of serine, threonine, or tyrosine amino acids in proteins.
Enzymes that mediate phosphorylation of cellular effectors generally fall into two classes. The first class consists of protein kinases which transfer a phosphate moiety from adenosine triphosphate to protein substrates. The second class consists of protein phosphatases which hydrolyze phosphate moieties from phosphoryl protein substrates. The converse functions of protein kinases and protein phosphatases balance and regulate the flow of signals in signal transduction processes.
Protein kinases and protein phosphatases are generally divided into two groupsxe2x80x94receptor and non-receptor type proteins. Most receptor-type protein tyrosine phosphatases contain two conserved catalytic domains, each of which encompasses a segment of 240 amino acid residues. Saito et al., 1991, Cell Growth and Diff. 2:59-65. Receptor protein tyrosine phosphatases can be subclassified further based upon the amino acid sequence diversity of their extracellular domains. Saito et al., supra; Krueger et al., 1992, Proc. Natl. Acad. Sci. USA 89:7417-7421.
Protein kinases and protein phosphatases are also typically divided into three classes based upon the amino acids they act upon. Some catalyze the addition or hydrolysis of phosphate on serine or threonine only, some catalyze the addition or hydrolysis of phosphate on tyrosine only, and some catalyze the addition or hydrolysis of phosphate on serine, threonine, and tyrosine.
Tyrosine kinases can regulate the catalytic activity of other protein kinases involved in cell proliferation. Protein kinases with inappropriate activity are also involved in some types of cancer. Abnormally elevated levels of cell proliferation are associated with receptor and non-receptor protein kinases with unregulated activity.
In addition to their role in cellular proliferation, protein kinases are thought to be involved in cellular differentiation processes. Cell differentiation occurs in some cells upon nerve growth factor (NGF) or epidermal growth factor (EGF) stimulation. Cellular differentiation is characterized by rapid membrane ruffling, cell flattening, and increases in cell adhesion. Chao, 1992, Cell 68:995-997.
In an effort to discover novel treatments for cancer and other diseases, biomedical researchers and chemists have designed, synthesized, and tested molecules that inhibit the function of protein kinases. Some small organic molecules form a class of compounds that modulate the function of protein kinases. Examples of molecules that have been reported to inhibit the function of protein kinases are bis-monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642), vinylene-azaindole derivatives (PCT WO 94/14808), 1-cyclopropyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992), styryl compounds (by Levitzki, et al., U.S. Pat. No. 5,217,999, and entitled xe2x80x9cStyryl Compounds which Inhibit EGF Receptor Protein Tyrosine Kinase, Lyon and Lyon), styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives (EP Application No. 0 566 266 A1), seleoindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660), and benzylphosphonic acid compounds (PCT WO 91/15495).
The compounds that can traverse cell membranes and are resistant to acid hydrolysis are potentially advantageous therapeutics as they can become highly bioavailable after being administered orally to patients. However, many of these protein kinase inhibitors only weakly inhibit the function of protein kinases. In addition, many inhibit a variety of protein kinases and will therefore cause multiple side-effects as therapeutics for diseases.
Despite the significant progress that has been made in developing compounds for the treatment of cancer, there remains a need in the art to identify the particular structures and substitution patterns that form the compounds capable of modulating the function of particular protein kinases.
The present invention is directed in part towards indolinone compounds and methods of modulating the function of protein tyrosine kinases with the indolinone compounds. The methods incorporate cells that express protein tyrosine kinases. In addition, the invention describes methods of preventing and treating protein tyrosine kinases-related abnormal conditions in organisms with a compound identified by the methods described herein. Furthermore, the invention pertains to pharmaceutical compositions comprising compounds identified by methods of the invention.
Thus, in a first aspect, the invention provides an indolinone compound having a structure set forth in formula I: 
where
(A) Q is an oxindole moiety having the structure set forth in formula II; 
xe2x80x83where
(a) R1, R2, and R3 are independently selected from the group consisting of
(i) hydrogen;
(ii) saturated or unsaturated alkyl optionally substituted with halogen, trihalomethyl, carboxylate, nitro, ester, and a five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moiety, where the ring moiety is optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester moieties;
(iii) an aromatic or heteroaromatic ring optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester moieties,
(iv) an aliphatic or heteroaliphatic ring optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, ester, and an aromatic or heteroaromatic ring optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester moieties;
(v) an amine of formula xe2x80x94(X1)n1xe2x80x94NX2X3, where X1 is selected from the group consisting of saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, or aliphatic ring moieties and where n1 is 0 or 1, and where X2 and X3 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, or aliphatic ring moieties;
(vi) a nitro of formula xe2x80x94NO2;
(vii) a halogen or trihalomethyl;
(viii) a ketone of formula xe2x80x94(X4)n4xe2x80x94COxe2x80x94X5 where X4 and X5 are independently selected from the group consisting of alkyl optionally substituted with halogen, trihalomethyl, carboxylate, nitro, ester, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring moieties are optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester moieties and where n4 is 0 or 1;
(ix) a carboxylic acid of formula xe2x80x94(X6)n6xe2x80x94COOH or ester of formula xe2x80x94(X7)n7xe2x80x94COOxe2x80x94X8, where X6, X7, and X8 and are independently selected from the group consisting of alkyl and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties and where n6 and n7 are independently 0 or 1;
(x) an alcohol of formula xe2x80x94(X9)n9xe2x80x94OH or an alkoxyalkyl moiety of formula xe2x80x94(X10)n10xe2x80x94Oxe2x80x94X11, where X9, X10, and X11 are independently selected from the group consisting of saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n9 and n10 are independently 0 or 1;
(xi) an amide of formula xe2x80x94(X12)n12xe2x80x94NHCOX13, or of formula xe2x80x94(X14)n14xe2x80x94CONX15X16, where X12 and X14 are each independently selected from the group consisting of alkyl, hydroxyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n12 and n14 are independently 0 or 1, and where X13, X15, and X16 are each independently selected from the group consisting of hydrogen, alkyl, hydroxyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester;
(xii) a sulfonamide of formula xe2x80x94(X17)n17xe2x80x94SO2NX18X19, where X17 is selected from the group consisting of hydrogen, alkyl, five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, or ester, and where X18, and X19 are independently selected from the group consisting of hydrogen, alkyl, five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, or ester, and where X18 and X19 taken together form a five-membered or six-membered aliphatic or heteroaliphatic ring optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n17 is 0 or 1;
(xiii) an aldehyde of formula xe2x80x94(X20)n20xe2x80x94COxe2x80x94H where X20 is selected from the group consisting of saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n20 is 0 or 1; and
(xiv) a sulfone of formula xe2x80x94(X21)n21xe2x80x94SO2xe2x80x94X22, where X22 is selected from the group consisting of hydroxide, saturated or unsaturated alkyl and five-membered or six-membered aryl or heteroaryl moieties and where X21 is selected from the group consisting of saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n21 is 0 or 1, and
(xv) a thiol of formula xe2x80x94(X23)n23xe2x80x94SH or a thioether of formula xe2x80x94(X24)n24xe2x80x94Sxe2x80x94X25, where X23, X24, and X25 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n23 and n24 are independently 0 or 1;
(b) A, B, D, and E are selected from the group consisting of carbon and nitrogen; and
(c) Q is bonded with the rest of the molecule through position 3 of the oxindole ring, as set forth in formula II; and
(B) T is a ring moiety having the structure set forth in formula III: 
xe2x80x83where
(a) R4, R5, R6, and R7 are independently selected from the group consisting of
(i) hydrogen;
(ii) saturated or unsaturated alkyl optionally substituted with halogen, trihalomethyl, carboxylate, nitro, ester, and a five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moiety, where the ring moiety is optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester moieties;
(iii) an aromatic or heteroaromatic ring optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester moieties;
(iv) an aliphatic or heteroaliphatic ring optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, ester, and an aromatic or heteroaromatic ring optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester moieties;
(v) an amine of formula xe2x80x94(X1)n1xe2x80x94NX2X3, where X1 is selected from the group consisting of saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, or aliphatic ring moieties and where n1 is 0 or 1, and where X2 and X3 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, or aliphatic ring moieties;
(vi) a nitro of formula xe2x80x94NO2;
(vii) a halogen or trihalomethyl;
(viii) a ketone of formula xe2x80x94(X4)n4xe2x80x94COxe2x80x94X5, where X4 and X5 are independently selected from the group consisting of alkyl optionally substituted with halogen, trihalomethyl, carboxylate, nitro, ester, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring moieties are optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro and ester moieties and where n4 is 0 or 1;
(ix) a carboxylic acid of formula xe2x80x94(X6)n6xe2x80x94COOH or ester of formula xe2x80x94(X7)n7xe2x80x94COOxe2x80x94X8, where X6, X7, and X8 and are independently selected from the group consisting of alkyl and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties and where n6 and n7 are independently 0 or 1;
(x) an alcohol of formula xe2x80x94(X9)n9xe2x80x94OH or an alkoxyalkyl moiety of formula xe2x80x94(X10)n10xe2x80x94Oxe2x80x94X11, where X9, X10, and X11 are independently selected from the group consisting of saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n9 and n10 are independently 0 or 1;
(xi) an amide of formula xe2x80x94(X12)n12xe2x80x94NHCOX13, or of formula xe2x80x94(X14)n14xe2x80x94CONX15X16, where X12 and X14 are each independently selected from the group consisting of alkyl, hydroxyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n12 and n14 are independently 0 or 1, and where X13, X15, and X16 are each independently selected from the group consisting of hydrogen, alkyl, hydroxyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester;
(xii) a sulfonamide of formula xe2x80x94(X17)n17xe2x80x94SO2NX18X19, where X17 is selected from the group consisting of alkyl, five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, or ester, and where X18, and X19 are independently selected from the group consisting of hydrogen, alkyl, five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, or ester, and where X18 and X19 taken together form a five-membered or six-membered aliphatic or heteroaliphatic ring optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n17 is 0 or 1;
(xiii) an aldehyde of formula xe2x80x94(X20)n20xe2x80x94COxe2x80x94H where X20 is selected from the group consisting of saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n20 is 0 or 1; and
(xiv) a sulfone of formula xe2x80x94(X21)n21xe2x80x94SO2xe2x80x94X22, where X22 is selected from the group consisting of hydroxide, saturated or unsaturated alkyl and five-membered or six-membered aryl or heteroaryl moieties and where X21 is selected from the group consisting of saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n21 is 0 or 1; and
(xv) a thiol of formula xe2x80x94(X23)n23xe2x80x94SH or a thioether of formula xe2x80x94(X24)n24xe2x80x94Sxe2x80x94X25, where X23, X24, and X25, are independently selected from the group consisting of saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n23 and n24 are independently 0 or 1;
(b) X is selected from the group consisting of NX26, sulfur, SO, SO2, and oxygen, where X26 is selected from tho group consisting of
(i) hydrogen;
(ii) saturated or unsaturated alkyl optionally substituted with a five-membered or six-membered aryl or heteroaryl ring moiety, where the ring moiety is optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester moieties; p3 (iii) an aryl optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester moieties;
(iv) a sulfone of formula xe2x80x94SO2xe2x80x94X27, where X27 is selected from the group consisting of saturated or unsaturated alkyl and five-membered or six-membered aryl or heteroaryl moieties; and
(v) an acyl of formula xe2x80x94C(O)X28, where X28 is selected from the group consisting of hydrogen, saturated and unsaturated alkyl, aryl, and a five-membered or six-membered ring moiety;
c) ring Y is selected from the group consisting of five-membered, six-membered, and seven-membered aromatic, heteroaromatic, or non-aromatic rings, where the heteroaromatic ring contains a heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur, and where the non-aromatic ring in combination with R4 optionally forms a carbonyl functionality;
(d) G, J, and L are selected from the group consisting of carbon and nitrogen; and
(e) T is bonded with the rest of the molecule through position of the ring marked with an asterisk (*), as get forth in formula III.
The term xe2x80x9cindolinonexe2x80x9d is used as that term is commonly understood in the art and includes a large subclass of substituted or unsubstituted of compounds that are capable of being synthesized from an aldehyde moiety and a oxindole moiety.
The term xe2x80x9coxindolexe2x80x9d refers to an oxindole compound substituted with chemical substituents. Oxindole compounds are of the general structure: 
The term xe2x80x9csubstitutedxe2x80x9d, in reference to the invention, refers to an oxindole compound that is derivatized with any number of chemical substituents.
The term xe2x80x9csaturated alkylxe2x80x9d refers to an alkyl moiety that does not contain any alkene or alkyne moieties. The alkyl moiety may be branched or non-branched.
The term xe2x80x9cunsaturated alkylxe2x80x9d refers to an alkyl moiety that contains at least one alkene or alkyne moiety. The alkyl moiety may be branched or non-branched.
The term xe2x80x9calkylenexe2x80x9d refers to a chain, either strait or branched, CH2 groups where the chain at both ends is attached to another functional group. Thus, for example, methylene refers to xe2x80x94CH2xe2x80x94, ethylene refers to xe2x80x94CH2CH2xe2x80x94, and propylene refers to xe2x80x94CH2CH2CH2xe2x80x94.
The term xe2x80x9caromaticxe2x80x9d refers to an aromatic group which has at least one ring having a conjugated pi electron system and includes both carbocyclic aryl (e.g. phenyl) and heterocyclic aryl groups (e.g. pyridine). The term xe2x80x9ccarbocyclicxe2x80x9d refers to a compound which contains one or more covalently closed ring structures, and that the atoms forming the backbone of the ring are all carbon atoms. The term thus distinguishes carbocyclic from heterocyclic rings in which the ring backbone contains at least one atom which is different from carbon. The term xe2x80x9cheteroaromaticxe2x80x9d refers to an aromatic group which contains at least one heterocyclic ring.
The term xe2x80x9caliphatic ringxe2x80x9d refers to a compound which contains one or more covalently closed ring structures, and that at least one of the atoms forming the backbone is a saturated carbon atom (e.g. cyclohexane). The term xe2x80x9cheteroaliphatic ringxe2x80x9d refers to a ring system in which at least one of the atoms forming the backbone is a heteroatom (e.g. tetrahydropyran).
The term xe2x80x9caminexe2x80x9d refers to a chemical moiety of formula NR1R2 where R1 and R2 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and five-membered or six-membered aryl or heteroaryl ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester moieties.
The term xe2x80x9chalogenxe2x80x9d refers to an atom selected from the group consisting of fluorine, chlorine, bromine, and iodine. The term xe2x80x9ctrihalomethylxe2x80x9d refers to the xe2x80x94CX3 group, where X is a halogen.
The term xe2x80x9cketonexe2x80x9d refers to a chemical moiety with formula xe2x80x94(R)nxe2x80x94COxe2x80x94Rxe2x80x2, where R and Rxe2x80x2 are selected from the group consisting of saturated or unsaturated alkyl and five-membered or six-membered aryl or heteroaryl moieties and where n is 0 or 1.
The term xe2x80x9ccarboxylic acidxe2x80x9d refers to a chemical moiety with formula xe2x80x94(R)nxe2x80x94COOH, where R is selected from the group consisting of saturated or unsaturated alkyl and five-membered or six-membered aryl or heteroaryl moieties and where n is 0 or 1. The term xe2x80x9ccarboxylatexe2x80x9d, in the context of this invention, refers to both a carboxylic acid and its anion, xe2x80x94(R)nxe2x80x94COOxe2x88x92.
The term xe2x80x9cesterxe2x80x9d refers to a chemical moiety with formula xe2x80x94(R)nxe2x80x94COORxe2x80x2, where R and Rxe2x80x2 are independently selected from the group consisting of saturated or unsaturated alkyl and five-membered or six-membered aryl or heteroaryl moieties and where n is 0 or 1.
The term xe2x80x9calcoholxe2x80x9d refers to a chemical substituent of formula xe2x80x94ROH, where R is selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and five-membered or six-membered aryl or heteroaryl ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester moieties.
The term xe2x80x9calkoxyalkyl moietyxe2x80x9d refers to a chemical substituent of formula xe2x80x94(R)nxe2x80x94ORxe2x80x2, where Rxe2x80x2 is an optionally substituted saturated or unsaturated alkyl moiety or an optionally substituted ring and n is 0 or 1, and where Rxe2x80x2 is an optionally substituted alkyl or optionally substituted aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties. When n is 0, then the alkoxyalkyl moiety is called an xe2x80x9calkoxy moietyxe2x80x9d.
The term xe2x80x9camidexe2x80x9d refers to a chemical substituent of formula xe2x80x94NHCOR, where R is selected from the group consisting of hydrogen, alkyl, hydroxyl, and five-membered or six-membered aryl or heteroaryl ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, or ester.
The term xe2x80x9caldehydexe2x80x9d refers to a chemical moiety with formula xe2x80x94(R)nxe2x80x94CHO, a where R is selected from the group consisting of saturated or unsaturated alkyl and five-membered or six-membered aryl or heteroaryl moieties and where n is 0 or 1.
The term xe2x80x9csulfonexe2x80x9d refers to a chemical moiety with formula xe2x80x94SO2xe2x80x94R, where R is selected from the group consisting of saturated or unsaturated alkyl and five-membered or six-membered aryl or heteroaryl moieties.
The term xe2x80x9cthiolxe2x80x9d refers to a chemical moiety with formula xe2x80x94(R)nxe2x80x94SH, where R is selected from the group consisting of optionally substituted alkyl or optionally substituted aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties and where n is 0 or 1. The term xe2x80x9cthioetherxe2x80x9d refers to a chemical moiety of the formula xe2x80x94(R)nxe2x80x94SRxe2x80x2 where both R and Rxe2x80x2 are selected from the group consisting of optionally substituted alkyl or optionally substituted aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties and where n is 0 or 1.
The term xe2x80x9cacylxe2x80x9d refers to chemical moieties of the general formula xe2x80x94C(O)R. When R is hydrogen the molecule containing the acyl group is an aldehyde. When R is an alkyl, an aliphatic ring, or an aromatic ring, then the molecule containing the acyl group is a ketone.
In preferred embodiments, the invention relates to an indolinone compound of formula I, where R1 and R2 are selected from the group consisting of
(i) hydrogen;
(ii) saturated alkyl optionally substituted with halogen, trihalomethyl, carboxylate, nitro, ester, and an aliphatic or heteroaliphatic ring optionally substituted with halogen, trihalomethyl, carboxylate, nitro, and ester moieties;
(iii) an aromatic ring optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, and nitro moieties;
(iv) an amine of formula xe2x80x94(X1)n1xe2x80x94NX2X3, where X1 is optionally substituted saturated alkyl and where n1 is 0 or 1, and where X2, and X3 are independently selected from the group consisting of hydrogen and optionally substituted saturated alkyl;
(v) a nitro of formula xe2x80x94NO2;
(vi) a halogen or trihalomethyl;
(vii) a ketone of formula xe2x80x94(X4)n4xe2x80x94COxe2x80x94X5, where X4 and X5 are alkyl and where n4 is 0 or 1;
(viii) a carboxylic acid of formula xe2x80x94(X6)n6xe2x80x94COOH or ester of formula xe2x80x94(X7)n7xe2x80x94COOxe2x80x94X8, where X6, X7, and X8 are alkyl and where n6 and n7 are independently 0 or 1;
(ix) an alcohol of formula xe2x80x94(X9)n9xe2x80x94OH or an alkoxyalkyl moiety of formula xe2x80x94(X10)n10xe2x80x94Oxe2x80x94X11, where X9, X10, and X11 are saturated alkyl and where n9 and n10 are independently 0 or 1;
(x) an amide of formula xe2x80x94(X12)n12xe2x80x94NHCOX13, or of formula xe2x80x94(X14)n14xe2x80x94CONX15X16, where X12, X13, X14, X15, and X16 are each independently selected from the group consisting of alkyl ad five-membered or six-membered aromatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, carboxylate, or ester and where n12 and n14 are independently 0 or 1; and
(xi) a sulfonamide of formula xe2x80x94(X17)n17xe2x80x94SO2NX18X19, where X17 is selected from the group consisting of alkyl, five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, or ester, and where X18 and X19 are independently selected from the group consisting of hydrogen, alkyl, five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, or ester, and where X18 and X19 taken together form a five-membered or six-membered aliphatic or heteroaliphatic ring optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n17 is 0 or 1.
More preferably, R1 and R2 of the indolinone of the invention are selected from the group consisting of
(i) hydrogen;
(ii) methyl, ethyl, propyl, and butyl groups optionally substituted with halogen, trihalomethyl, cyano, and nitro moieties;
(iii) phenyl optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, and nitro moieties;
(iv) an amine of formula xe2x80x94(X1)n1xe2x80x94NX2X3, where X2 and X3 are independently selected from the group consisting of hydrogen and optionally subsituted saturated alkyl, and X1 is optionally substituted saturated alkyl, and where n is 0 or 1;
(v) a nitro of formula xe2x80x94NO2;
(vi) a halogen or trihalomethyl;
(vii) a ketone of formula xe2x80x94COxe2x80x94X4, where X4 is selected from the group consisting of methyl, ethyl, propyl, and butyl;
(viii) a carboxylic acid of formula xe2x80x94(X6)n6xe2x80x94COOH or ester of formula xe2x80x94(X7)n7xe2x80x94COOxe2x80x94X8, where X6 and X7 are selected from the group consisting of a bond, methylene, ethylene, and propylene, and where X8 is selected from the group consisting of methyl and ethyl, and where n6 and n7 are independently 0 and 1;
(ix) an alkoxy moiety of formula xe2x80x94Oxe2x80x94X11, where X11 is selected from the group consisting of methyl and ethyl;
(x) an amide of formula xe2x80x94NHCOX13, where X13 is phenyl optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, carboxylate, or ester; and
(xi) a sulfonamide of formula xe2x80x94SO2NX18X19, where X18 and X19 are independently selected from the group consisting of hydrogen, methyl, ethyl, phenyl optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, and trihalomethyl, and where X18 and X19 taken together form a six-membered heteroaliphatic ring moiety.
In some embodiments, E in formula II is nitrogen.
Most preferably, the indolinone compounds of the invention are those whose structure is set forth in formula I, where Q is selected from the group consisting of type Q oxindoles. By xe2x80x9ctype Q oxindolesxe2x80x9d is meant oxindole compounds which are selected from the list of oxindoles numbered O-1 through O-60, and are depicted as follows. 
In certain other preferred embodiments, the indolinone of the invention has a structure as set forth in formula I, where R4 and R5 are independently selected from the group consisting of
(i) hydrogen;
(ii) methyl, ethyl, propyl, and butyl groups optionally substituted with halogen, trihalomethyl, cyano, and nitro moieties;
(iii) an amine of formula xe2x80x94(X1)n1xe2x80x94NX2X3, where X2 and X3 are independently selected from the group consisting of hydrogen and substituted saturated alkyl, and X1 is substituted saturated alkyl, and where n1 is 0 or 1, or where X2 and X3 taken together form a five-membered or a six-membered aliphatic or heteroaliphatic ring, optionally substituted at a ring carbon atom or heteroatom with a substituent selected from the group consisting of methyl, ethyl, propyl, phenyl, and alkoxyphenyl;
(iv) a nitro of formula xe2x80x94NO2;
(v) a halogen or trihalomethyl;
(vi) a ketone of formula xe2x80x94COxe2x80x94X4, where X4 is selected from the group consisting of methyl, ethyl, propyl, n-butyl, and t-butyl;
(vii) a carboxylic acid of formula xe2x80x94(X6)n6xe2x80x94COOH or ester of formula xe2x80x94(X7)n7xe2x80x94COOxe2x80x94X8, where X6 and X7 are selected from the group consisting of a bond, methylene, ethylene, and propylene, and where X8 is selected from the group consisting of methyl and ethyl, and where n6 and n7 are independently 0 or 1;
(viii) an amide of formula xe2x80x94NHCOX13, or of formula xe2x80x94CONX15X16, where X13, X15, and X16 are each independently selected from the group consisting of hydrogen, methyl, ethyl, propyl, and phenyl;
(ix) a sulfonamide of formula xe2x80x94SO2NX18X19, where X18 and X19 are independently selected from the group consisting of hydrogen, methyl, and ethyl;
(x) an alcohol of formula xe2x80x94(X9)n9xe2x80x94OH or an alkoxyalkyl moiety of formula xe2x80x94(X10)n10xe2x80x94Oxe2x80x94X11, where X9, and X10 are independently selected form the group consisting of methylene, ethylene, and propylene, and where X11 is independently selected from the group consisting of methyl, ethyl, and propyl, and where n9 and n10 are independently 0 or 1;
(xi) a sulfone of formula xe2x80x94(X21)n21xe2x80x94SO2xe2x80x94X22, where X22 is selected from the group consisting of hydroxide, saturated or unsaturated alkyl, and five-membered or six-membered aryl or heteroaryl moieties, and where X21 is saturated alkyl, and where n21 is 0 or 1; and
(x) a thioether of formula xe2x80x94(X24)n24xe2x80x94Sxe2x80x94X25, where X24 is independently selected from the group consisting of methylene, ethylene, and propylene, and where X25 is independently selected from the group consisting of methyl, ethyl, propyl, and phenyl, and where n24 is 0 or 1.
More preferably, R4 and R5 are each independently selected from the group consisting of
(i) hydrogen;
(ii) methyl and ethyl;
(iii) an amine of formula xe2x80x94(X1)n1xe2x80x94NX2X3, where X2 and X3 are independently selected from the group consisting of hydrogen methyl and ethyl, and X1 is methylene or ethylene, and where n1 is 0 or 1, or where X2 and X3 taken together form a substituted ring selected from the group consisting of 
(iv) a nitro of formula xe2x80x94NO2;
(v) a halogen;
(vi) a ketone of formula xe2x80x94COxe2x80x94X4, where X4 is selected from the group consisting of methyl and t-butyl;
(vii) a carboxylic acid of formula xe2x80x94(X6)n6xe2x80x94COOH or ester of formula xe2x80x94(X7)n7xe2x80x94COOxe2x80x94X8, where X6 and X7 are selected from the group consisting of a bond, methylene, ethylene, and propylene, and where X8 is selected from the group consisting of methyl and ethyl, and where n6 and n7 are independently 0 or 1;
(viii) an amide of formula xe2x80x94NHCOX13, or of formula xe2x80x94CONX15X16, where X13, X15, and X16 are each independently selected from the group consisting of hydrogen, methyl, and phenyl;
(ix) a sulfonamide of formula xe2x80x94SO2NX18X19, where X18 and X19 are independently selected from the group consisting of hydrogen, methyl, and ethyl;
(x) an alcohol of formula xe2x80x94(X9)n9xe2x80x94OH or an alkoxyalkyl moiety of formula xe2x80x94(X10)n10xe2x80x94Oxe2x80x94X11, where X9, and X10 are independently selected form the group consisting of methylene, ethylene, and propylene, and where X11 is independently selected from the group consisting of methyl, ethyl, and propyl, and where n9 and n10 are independently 0 or 1;
(xi) a sulfone of formula xe2x80x94SO2xe2x80x94X22, where X22 is hydroxide; and
(xi) a thioether of formula xe2x80x94Sxe2x80x94X25, where X25 is phenyl.
Preferably, R6 and R7 groups of the indolinone compounds of the invention are independently selected from the group consisting of
(i) hydrogen;
(ii) methyl, ethyl, propyl, and butyl groups optionally substituted with halogen, trihalomethyl, cyano, and nitro moieties;
(iii) an amine of formula xe2x80x94(X1)n1xe2x80x94NX2X3, where X2 and X3 are independently selected from the group consisting of hydrogen and substituted saturated alkyl, and X1 is an optionally substituted saturated alkylene, and where n1 is 0 or 1;
(iv) a halogen or trihalomethyl;
(v) an alcohol of formula xe2x80x94(X9)n9xe2x80x94OH or an alkoxyalkyl moiety of formula xe2x80x94(X10)n10xe2x80x94Oxe2x80x94X11, where X9 and X10 are independently selected form the group consisting of methylene, ethylene, and propylene, and where X11 is independently selected form the group consisting of methyl, ethyl, and propyl, and where n9 and n10 are independently 0 or 1.
More preferably, R6 and R7 are independently selected from the group consisting of
(i) hydrogen;
(ii) methyl and ethyl,
(iii) an amine of formula xe2x80x94(X1)n1xe2x80x94NX2X3, where X2 and X3 are independently selected from the group consisting of hydrogen, methyl, and ethyl, and X1 is selected from the group consisting of methylene and ethylene, and where n1 is 0 or 1;
(iv) a halogen;
(v) a hydroxy xe2x80x94OH or an alkoxy moiety of formula xe2x80x94Oxe2x80x94X11, where X11 is independently selected form the group consisting of methyl, ethyl, and propyl.
Preferably, the Y ring of the indolinone compound of the invention is a six-membered aromatic or heteroaromatic ring. If Y is an aromatic ring, then the moiety set forth in formula III would take the form set forth in formula V 
with the rings optionally substituted as described herein and E having the limitations set forth herein. If Y is a heteroaromatic ring, then at least one of carbon atoms of the Y ring of the structure set forth in formula V is a heteroatom (e.g. nitrogen).
In some other preferred embodiments, the Y ring of the indolinone compounds is a six-membered aliphatic or heteroaliphatic ring. Then, the Y ring in the structure set forth in formula V takes the form of, for example, optionally substituted 
In other preferred embodiments, G, J, and L are independently nitrogen. X may also preferably be oxygen, nitrogen, optionally substituted with an alkyl, or be selected from the group consisting of sulfur, SO, and SO2.
In preferred embodiments, the precursor to the T moiety of the indolinone compound of the invention set forth in formula I is selected from the group consisting of type T aldehydes. By xe2x80x9ctype T aldehydexe2x80x9d is meant aldehyde compounds which are selected from the list of aldehydes numbered A-1 through A-95, and are depicted as follows. 
In another aspect, the invention relates to a combinatorial library of at least 10 indolinone compounds that can be formed by reacting oxindoles with aldehydes. In preferred embodiments, the oxindoles are those that have a structure set forth in formula II as defined herein or any of the subgroups thereof set forth herein. The oxindoles ale preferably selected from the group consisting of type Q oxindoles.
The aldehydes of the combinatorial library of the invention preferably have a structure set forth in formula IV: 
with R4, R5, R6, R7, G, J, L, X, and Y are as defined herein for the compound of formula III, or any of the subgroups thereof set forth herein. Preferably, the aldehydes are selected from the group consisting of type T aldehydes.
In a further aspect, the invention features a method for synthesizing an indolinone compound comprising the steps of:
(a) reacting a first reactant with a second reactant in a solvent and in the presence of a base at elevated temperatures, where the first reactant is an oxindole, and where the second reactant is an aldehyde;
(b) purifying the indolinone compound.
The first reactant is preferably selected from the group consisting of type Q oxindoles and the second reactant is preferably selected from the group consisting of type T aldehydes. The base is preferably a nitrogen base, and most preferably, the base is piperidine.
xe2x80x9cNitrogen basesxe2x80x9d are commonly used in the art and are selected from acyclic and cyclic amines. Examples of nitrogen bases include, but are not limited to, ammonia, methyl amine, trimethylamine, aniline, and piperidine. Those skilled in the art know which nitrogen base would match the requirements of the reaction conditions.
The solvent of the reaction is preferably an alcohol, and most preferably, the solvent is ethanol.
The synthetic method of the invention calls for the reaction to take place at elevated temperatures. The term xe2x80x9celevated temperaturesxe2x80x9d refers to temperatures that are greater than room temperature. More preferably, the elevated temperature is about 90xc2x0 C. By xe2x80x9cabout 90xc2x0 C.xe2x80x9d it is meant that the temperature range is preferably 90xc2x110xc2x0 C., more preferably 90xc2x15xc2x0 C., and most preferably 90xc2x12xc2x0 C.
In another aspect, the invention features a pharmaceutical composition comprising (i) a physiologically acceptable carrier, diluent, or excipient; and (ii) an indolinone compound as described herein, or a salt thereof.
The tenn xe2x80x9cpharmaceutical compositionxe2x80x9d refers to a mixture of an indolinone compound of the invention with other chemical components, such as diluents, excipients, or carriers. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, oral, injection, aerosol, parenteral, and topical administration. Pharmaceutical compositions can also be obtained by reacting compounds with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
The term xe2x80x9cphysiologically acceptablexe2x80x9d defines a carrier or diluent that does not abrogate the biological activity and properties of the compound.
The term xe2x80x9ccarrierxe2x80x9d defines a chemical compound that facilitates the incorporation of a compound into cells or tissues. For example dimethyl sulfoxide (DMSO) is a commonly utilized carrier as it facilitates the uptake of many organic compounds into the cells or tissues of an organism.
The term xe2x80x9cdiluentxe2x80x9d defines chemical compounds diluted in water that will dissolve the compound of interest as well as stabilize the biologically active form of the compound. Salts dissolved in buffered solutions are utilized as diluents in the art. One commonly used buffered solution is phosphate buffered saline because it mimics the salt conditions of human blood. Since buffer salts can control the pH of a solution at low concentrations, a buffered diluent rarely modifies the biological activity of a compound.
The invention also features a method of modulating the function of a protein tyrosine kinase with an indolinone compound, comprising the step of contacting cells expressing the protein tyrosine kinase with the compound.
The term xe2x80x9cfunctionxe2x80x9d refers to the cellular role of a protein tyrosine kinase. The protein tyrosine kinase family includes members that regulate many steps in signaling cascades, including cascades controlling cell growth, migration, differentiation, gene expression, muscle contraction, glucose metabolism, cellular protein synthesis, and regulation of the cell cycle.
The term xe2x80x9ccatalytic activityxe2x80x9d, in the context of the invention, defines the rate at which a protein kinase phosphorylates a substrate. Catalytic activity can be measured, for example, by determining the amount of a substrate converted to a product as a function of time. Phosphorylation of a substrate occurs at the active-site of a protein kinase. The active-site is normally a cavity in which the substrate binds to the protein kinase and is phosphorylated.
The term xe2x80x9csubstratexe2x80x9d as used herein refers to a molecule phosphorylated by a protein tyrosine kinase. The substrate is preferably a peptide and more preferably a protein.
The term xe2x80x9cactivatesxe2x80x9d refers to increasing the cellular function of a protein kinase. The protein kinase unction is preferably the interaction with a natural binding partner and most preferably catalytic activity.
The term xe2x80x9cinhibitxe2x80x9d refers to decreasing the cellular function of a protein kinase. The protein kinase function is preferably the interaction with a natural binding partner and most preferably catalytic activity.
The term xe2x80x9cmodulatesxe2x80x9d refers to altering the function of a protein kinase by increasing or decreasing the probability that a complex forms between a protein kinase and a natural binding partner. A modulator preferably increases the probability that such a complex forms between the protein kinase and the natural binding partner, more preferably increases or decreases the probability that a complex forms between the protein kinase and the natural binding partner depending on the concentration of the compound exposed to the protein kinase, and most preferably decreases the probability that a complex forms between the protein kinase and the natural binding partner. A modulator preferably activates the catalytic activity of a protein kinase, more preferably activates or inhibits the catalytic activity of a protein kinase depending on the concentration of the compound exposed to the protein kinase, or most preferably inhibits the catalytic activity of a protein kinase.
The term xe2x80x9ccomplexxe2x80x9d refers to an assembly of at least two molecules bound to one another. Signal transduction complexes often contain at least two protein molecules bound to one another.
The term xe2x80x9cnatural binding partnerxe2x80x9d refers to polypeptides that bind to a protein kinase in cells. Natural binding partners can play a role in propagating a signal in a protein kinase signal transduction process. A change in the interaction between a protein kinase and a natural binding partner can manifest itself as an increased or decreased probability that the interaction forms, or an increased or decreased concentration of the protein kinase/natural binding partner complex.
A protein kinase natural binding partner can bind to a protein kinase""s intracellular region with high affinity. High affinity represents an equilibrium binding constant on the order of 10xe2x88x926 M or less. In addition, a natural binding partner can also transiently interact with a protein kinase intracellular region and chemically modify it. Protein kinase natural binding partners are chosen from a group that includes, but is not limited to, SRC homology 2 (SH2) or 3 (SH3) domains, other phosphoryl tyrosine binding (PTB) domains, guanine nucleotide exchange factors, protein phosphatases, and other protein kinases. Methods of determining changes in interactions between protein kinases and their natural binding partners are readily available in the art.
The term xe2x80x9ccontactingxe2x80x9d as used herein refers to mixing a solution comprising an indolinone compound of the invention with a liquid medium bathing the cells of the methods. The solution comprising the compound may also comprise another component, such as dimethylsulfoxide (DMSO), which facilitates the uptake of the indolinone compound or compounds into the cells of the methods. The solution comprising the indolinone compound may be added to the medium bathing the cells by utilizing a delivery apparatus, such as a pipet-based device or syringe-based device.
The indolinone compounds of the invention preferably modulate the activity of the protein tyrosine kinase in vitro. These compounds preferably show positive results in one or more in vitro assays for an activity corresponding to treatment of the disease or disorder in question (such as the assays described in the Examples below). The protein tyrosine kinase which is modulated by the indolinone compounds of the invention is preferably HER2. The procedures for and the results of such modulation are described in the Examples below.
The invention also features a method of identifying indolinone compounds that modulate the function of protein tyrosine kinase, comprising the following steps: (a) contacting cells expressing the protein tyrosine kinase with the compound; and (b) monitoring an effect upon the cells. The effect upon the cells is preferably a change or an absence of a change in cell phenotype, more preferably it is a change or an absence of a change in cell proliferation, even more preferably it is a change or absence of a change in the catalytic activity of the protein tyrosine kinase, and most preferably it is a change or absence of a change in the interaction between the protein tyrosine kinase with a natural binding partner, as described herein.
The term xe2x80x9cmonitoringxe2x80x9d refers to observing the effect of adding the compound to the cells of the method. The effect can be manifested in a change in cell phenotype, cell proliferation, protein kinase catalytic activity, or in the interaction between a protein kinase and a natural binding partner.
The term xe2x80x9ceffectxe2x80x9d describes a change or an absence of a change in cell phenotype or cell proliferation. xe2x80x9cEffectxe2x80x9d can also describe a change or an absence of a change in the catalytic activity of the protein kinase. xe2x80x9cEffectxe2x80x9d can also describe a change or an absence of a change in an interaction between the protein kinase and a natural binding partner.
The term xe2x80x9ccell phenotypexe2x80x9d refers to the outward appearance of a cell or tissue or the function of the cell or tissue. Examples of cell phenotype are cell size (reduction or enlargement), cell proliferation (increased or decreased numbers of cells), cell differentiation (a change or absence of a change in cell shape), cell survival, apoptosis (cell death), or the utilization of a metabolic nutrient (e.g., glucose uptake). Changes or the absence of changes in cell phenotype are readily measured by techniques known in the art.
In a preferred embodiment, the invention features a method for identifying the indolinones of the invention, comprising the following steps: (a) lysing the cells to render a lysate comprising protein tyrosine kinase; (b) adsorbing the protein tyrosine kinase to an antibody; (c)incubating the adsorbed protein tyrosine kinase with a substrate or substrates, and (d) adsorbing the substrate or substrates to a solid support or antibody; where the step of monitoring the effect on the cells comprises measuring the phosphate concentration of the substrate or substrates.
The term xe2x80x9cantibodyxe2x80x9d refers to an antibody (e.g., a monoclonal or polyclonal antibody), or antibody fragment, having specific binding affinity to protein tyrosine kinase or its fragment.
By xe2x80x9cspecific binding affinityxe2x80x9d is meant that the antibody binds to target (protein tyrosine kinase) polypeptides with greater affinity than it binds to other polypeptides under specified conditions. Antibodies having specific binding affinity to a protein tyrosine kinase may be used in methods for detecting the presence and/or amount of a protein tyrosine kinase in a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount of the antibody conjugated to the protein tyrosine kinase. Diagnostic kits for performing such methods may be constructed to include a first container containing the antibody and a second container having a conjugate of a binding partner of the antibody and a label, such as, for example, a radioisotope. The diagnostic kit may also include notification of an FDA approved use and instructions therefor.
The term xe2x80x9cpolyclonalxe2x80x9d refers to antibodies that are heterogenous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof. For the production of polyclonal antibodies, various host animals may be immunized by injection with the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species.
xe2x80x9cMonoclonal antibodiesxe2x80x9d are substantially homogenous populations of antibodies to a particular antigen. They may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art. See, for example, Kohler, et al., Nature 256:495-497 (1975), and U.S. Pat. No. 4,376,110.
The term xe2x80x9cantibody fragmentxe2x80x9d refers to a portion of an antibody, often the hypervariable region and portions of the surrounding heavy and light chains, that displays specific binding affinity for a particular molecule. A hypervariable region is a portion of an antibody that physically binds to the polypeptide target.
In yet another aspect, the invention features a method for treating a disease related to unregulated tyrosine kinase signal transduction, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of an indolinone compound as described herein.
The invention also features a method of regulating tyrosine kinase signal transduction comprising administering to a subject a therapeutically effective amount of an indolinone compound as described herein.
Furthermore, the invention features a method of preventing or treating an abnormal condition in an organism, where the abnormal condition is associated with an aberration in a signal transduction pathway characterized by an interaction between a protein kinase and a natural binding partner, where the method comprises the following steps: (a) administering an indolinone compound as described herein; and (b) promoting or disrupting the abnormal interaction. The organism is preferably a mammal and the abnormal condition is preferably cancer. The abnormal condition may also preferably be selected from the group consisting of hypertension, depression, generalized anxiety disorder, phobias, post-traumatic stress syndrome, avoidant personality disorder, sexual dysfunction, eating disorders, obesity, chemical dependencies, cluster headache, migraine, pain, Alzheimer""s disease, obsessive-compulsive disorder, panic disorder, memory disorders, Parkinson""s disease, endocrine disorders, vasospasm, cerebellar ataxia, and gastrointestinal tract disorders.
The term xe2x80x9caberrationxe2x80x9d, in conjunction with a signal transduction process, refers to a protein kinase that is over- or under-expressed in an organism, mutated such that its catalytic activity is lower or higher than wild-type protein kinase activity, mutated such that it can no longer interact with a natural binding partner, is no longer modified by another protein kinase or protein phosphatase, or no longer interacts with a natural binding partner.
The term xe2x80x9cpromoting or disrupting the abnormal interactionxe2x80x9d refers to a method that can be accomplished by administering a compound of the invention to cells or tissues in an organism. A compound can promote an interaction between a protein kinase and natural binding partners by forming favorable interactions with multiple atoms at the complex interface. Alternatively, a compound can inhibit an interaction between a protein kinase and natural binding partners by compromising favorable interactions formed between atoms at the complex interface. The present invention also features novel oxindole compounds. In one aspect, the invention features an oxindole compound of formula V 
where R8 is selected from the group consisting of
(i) saturated alkyl, optionally substituted with a substituent selected from the group consisting of alkoxy, trihalomethyl, nitro, and cyano moieties, provided that the alkyl is not methyl;
(ii) an amine of formula xe2x80x94(X1)n1xe2x80x94NX2X3, where X1 is selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, or aliphatic ring moieties and where n1 is 0 or 1, and where X2 and X3 taken together form a 5-membered or 6-membered heteroaliphatic ring;
(iii) an iodine;
(iv) a ketone of formula xe2x80x94(X4)n4xe2x80x94COxe2x80x94X5, where X4 and X5 are independently alkyl and where n4 is 0 or 1;
(v) a carboxylic acid of formula xe2x80x94(X6)n6xe2x80x94COOH or ester of formula xe2x80x94(X7)n7xe2x80x94COOxe2x80x94X8 where X6, X7, and X8 and are independently selected from the group consisting of alkyl and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties and where n6 and n7 are independently 0 or 1;
(vi) an amide of formula xe2x80x94(X12)n12xe2x80x94NHCOX13, or of formula xe2x80x94(X14)n14xe2x80x94CONX15X16, where X12 and X14 are each independently selected from the group consisting of alkyl, hydroxyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n12 and n14 are independently 0 or 1, and where X13, X15, and X16 are each independently selected from the group consisting of hydrogen, alkyl, hydroxyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester; and
(vii) a sulfonamide of formula xe2x80x94(X17)n17xe2x80x94SO2NX18X19, where X17 is selected from the group consisting of hydrogen, alkyl, five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, or ester, and where X18, and X19 are independently selected from the group consisting of hydrogen, alkyl, five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, or ester, and where X18 and X19 taken together form a five-membered or six-membered aliphatic or heteroaliphatic ring optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n17 is 0 or 1.
The oxindole compound of formula V is preferably selected from the group consisting of 
In another aspect, the invention features an oxindole compound of formula VI 
where R9 is selected from the group consisting of
(i) an amine of formula xe2x80x94(X1)n1xe2x80x94NX2X3, where X1 is selected from the group consisting of saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, or aliphatic ring moieties and where n1 is 0 or 1, and where X2 and X3 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, or aliphatic ring moieties;
(ii) a nitro of formula xe2x80x94NO2;
(iii) a chlorine, bromine, or iodine;
(iv) a ketone of formula xe2x80x94(X4)n4xe2x80x94COxe2x80x94X5, where X4 and X5 are independently selected from the group consisting of alkyl optionally substituted with halogen, trihalomethyl, carboxylate, nitro, ester, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring moieties are optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester moieties and where n4 is 0 or 1;
(v) a carboxylic acid of formula xe2x80x94(X6)n6xe2x80x94COOH or ester of formula xe2x80x94(X7)n7xe2x80x94COOxe2x80x94X8, where X6, X7, and X8 and are independently selected from the group consisting of alkyl and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties and where n6 and n7 are independently 0 or 1;
(vi) an amide of formula xe2x80x94(X12)n12xe2x80x94NHCOX13, or of formula xe2x80x94(X14)n14xe2x80x94CONX15X16, where X12 and X14 are each independently selected from the group consisting of alkyl, hydroxyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n12 and n14 are independently 0 or 1, and where X13, X15, and X16 are each independently selected from the group consisting of hydrogen, alkyl, hydroxyl, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester; and
(vii) a sulfonamide of formula xe2x80x94(X17)n17xe2x80x94SO2NX18X19, where X17 is selected from the group consisting of hydrogen, alkyl, five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, or ester, and where X18, and X19 are independently selected from the group consisting of hydrogen, alkyl, five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, or ester, and where X18 and X19 taken together form a five-membered or six-membered aliphatic or heteroaliphatic ring optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n17 is 0 or 1.
The oxindole compound of formula VI is preferably selected from the group consisting of 
In another aspect, the invention features an oxindole compound of formula VII 
where R10 is selected from the group consisting of
(i) an aromatic or heteroaromatic ring optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, amide, and ester moieties;
(ii) an aliphatic or heteroaliphatic ring optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, ester, and an aromatic or heteroaromatic ring optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester moieties;
(iii) an amine of formula xe2x80x94(X1)n1xe2x80x94NX2X3, where X1 is selected from the group consisting of saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, or aliphatic ring moieties and where n1 is 0 or 1, and where X2 and X3 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and five-membered or six-membered aromatic, heteroaromatic, or aliphatic ring moieties, or where X2 and X3 taken together form a 5-membered or 6-membered heteroaliphatic ring;
(iv) a nitro of formula xe2x80x94NO2;
(v) a bromine;
(vi) a ketone of formula xe2x80x94(X4)n4xe2x80x94COxe2x80x94X5, where X4 and X5 are independently selected from the group consisting of alkyl optionally substituted with halogen, trihalomethyl, carboxylate, nitro, ester, and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties, where the ring moieties are optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester moieties and where n4 is 0 or 1;
(vii) a carboxylic acid of formula xe2x80x94(X6)n6xe2x80x94COOH or ester of formula xe2x80x94(X7)n7xe2x80x94COOxe2x80x94X8, where X6, X7, and X8 and are independently selected from the group consisting of alkyl and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties and where n6 and n7 are independently 0 or 1;
(viii) a sulfonamide of formula xe2x80x94(X17)n17xe2x80x94SO2NX18X9, where X17 is selected from the group consisting of hydrogen, alkyl, five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, or ester, and where X18, and X19 are independently selected from the group consisting of hydrogen, alkyl, five-membered or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, or ester, and where X18 and X19 taken together form a five-membered or six-membered aliphatic or heteroaliphatic ring optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester and where n17 is 0 or 1.
The oxindole compound of formula VII is preferably selected from the group consisting of 
In a further aspect, the invention features an oxindole compound, where the oxindole compound is selected from the group consisting of 
The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.
The present invention relates to compounds capable of regulating and/or modulating tyrosine kinase signal transduction and more particularly receptor and non-receptor tyrosine kinase signal transduction.
Receptor tyrosine kinase mediated signal transduction is initiated by extracellular interaction with a specific growth factor (ligand), followed by receptor dimerization, transient stimulation of the intrinsic protein tyrosine kinase activity and phosphorylation. Binding sites are thereby created for intracellular signal transduction molecules and lead to the formation of complexes with a spectrum of cytoplasmic signaling molecules that facilitate the appropriate cellular response (e.g., cell division, metabolic effects to the extracellular microenvironment). See, Schlessinger and Ullrich, 1992, Neuron 9:303-391.
It has been shown that tyrosine phosphorylation sites in growth factor receptors function as high-affinity binding sites for SH2 (src homology) domains of signaling molecules. Fantl et al., 1992, Cell 69:413-423; Songyang et al., 1994, Mol. Cell. Biol. 14:2777-2785); Songyang et al., 1993, Cell 72:767-778; and Koch et al., 1991, Science 252:668-678. Several intracellular substrate proteins that associate with receptor tyrosine kinases have been identified. They may be divided into two principal groups: (1) substrates which have a catalytic domain; and (2) substrates which lack such domain but serve as adapters and associate with catalytically active molecules. Songyang et al., 1993, Cell 72:767-778. The specificity of the interactions between receptors and SH2 domains of their substrates is determined by the amino acid residues immediately surrounding the phosphorylated tyrosine residue. Differences in the binding affinities between SH2 domains and the amino acid sequences surrounding the phosphotyrosine residues on particular receptors are consistent with the observed differences in their substrate phosphorylation profiles. Songyang et al., 1993, Cell 72:767-778. These observations suggest that the function of each receptor tyrosine kinase is determined not only by its pattern of expression and ligand availability but also by the array of downstream signal transduction pathways that are activated by a particular receptor. Thus, phosphorylation provides an important regulatory step which determines the selectivity of signaling pathways recruited by specific growth factor receptors, as well as differentiation factor receptors.
Tyrosine kinase signal transduction results in, among other responses, cell proliferation, differentiation and metabolism. Abnormal cell proliferation may result in a wide array of disorders and diseases, including the development of neoplasia such as carcinoma, sarcoma, leukemia, glioblastoma, hemangioma, psoriasis, arteriosclerosis, arthritis and diabetic retinopathy (or other disorders related to uncontrolled angiogenesis and/or vasculogenesis).
This invention is therefore directed to compounds which regulate, modulate and/or inhibit tyrosine kinase signal transduction by affecting the enzymatic activity of the RTKs and/or the non-receptor tyrosine kinases and interfering with the signal transduced by such proteins. More particularly, the present invention is directed to compounds which regulate, modulate and/or inhibit the RTK and/or non-receptor tyrosine kinase mediated signal transduction pathways as a therapeutic approach to cure many kinds of solid tumors, including but not limited to carcinoma, sarcoma, leukemia, erythroblastoma, glioblastoma, meningioma, astrocytoma, melanoma and myoblastoma. Indications may include, but are not limited to brain cancers, bladder cancers, ovarian cancers, gastric cancers, pancreatic cancers, colon cancers, blood cancers, lung cancers and bone cancers.
I. Target Diseases to be Treated by the Compounds of the Invention
The compounds described herein are useful for treating disorders related to unregulated tyrosine kinase signal transduction, including cell proliferative disorders, fibrotic disorders and metabolic disorders.
Cell proliferative disorders which can be treated or further studied by the present invention include cancers, blood vessel proliferative disorders and mesangial cell proliferative disorders.
Blood vessel proliferative disorders refer to angiogenic and vasculogenic disorders generally resulting in abnormal proliferation of blood vessels. The formation and spreading of blood vessels, or vasculogenesis and angiogenesis, respectively, play important roles in a variety of physiological processes such as embryonic development, corpus luteum formation, wound healing and organ regeneration. They also play a pivotal role in cancer development. Other examples of blood vessel proliferation disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilage, and ocular diseases, like diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness. Conversely, disorders related to the shrinkage, contraction or closing of blood vessels, such as restenosis, are also implicated.
Fibrotic disorders refer to the abnormal formation of extracellular matrix. Examples of fibrotic disorders include hepatic cirrhosis and mesangial cell proliferative disorders. Hepatic cirrohis is characterized by the increase in extracellular matrix constituents resulting in the formation of a hepatic scar. Hepatic cirrhosis can cause diseases such as cirrhosis of the liver. An increased extracellular matrix resulting in a hepatic scar can also be caused by viral infection such as hepatitis. Lipocytes appear to play a major role ion hepatic cirrhosis. Other fibrotic disorders implicated include atherosclerosis (see, below).
Mesangial cell proliferative disorders refer to disorders brought about by abnormal proliferation of mesangial cells. Mesangial proliferative disorders include various human renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes, transplant rejection, and glomerulopathies. The PDGF-R has been implicated in the maintenance of mesangial cell proliferation. Floege et al., 1993, Kidney International 43:47S-54S.
PTKs have been associated with such cell proliferative disorders. For example, some members of the RTK family have been associated with the development of cancer. Some of these receptors, like the EGFR (Tuzi et al, 1991, Br. J. Cancer 63:227-233; Torp et al., 1992, APMIS 100:713-719) HER2/neu (Slamon et al., 1989, Science 244:707-712) and the PDGF-R (Kumabe et al., 1992, Oncogene 7:627-633) are overexpressed in many tumors and/or persistently activated by autocrine loops. In fact, in the most common and severe cancers these receptor overexpressions (Akbasak and Suner-Akbasak et al., 1992, J. Neurol. Sci. 111:119-133; Dickson et al., 1992, Cancer Treatment Res. 61:249-273; Korc et al., 1992, J. Clin. Invest. 90:1352-1360) and autocrine loops (Lee and Donoghue, 1992, J. Cell. Biol. 118:1057-1070; Korc et al, supra; Akbasak and Suner-Akbasak et al., supra) have been demonstrated. For example, the EGFR receptor has been associated with squamous cell carcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancer and bladder cancer. HER2 has been associated with breast, ovarian, gastric, lung, pancreas and bladder cancer. The PDGF-R has been associated with glioblastoma, lung, ovarian, melanoma and prostate. The RTK c-met has been generally associated with hepatocarcinogenesis and thus hepatocellular carcinoma. Additionally, c-met has been linked to malignant tumor formation. More specifically, the RTK c-met has been associated with, among other cancers, colorectal, thyroid, pancreatic and gastric carcinoma, leukemia and lymphoma. Additionally, over-expression of the c-met gene has been detected in patients with Hodgkins disease, Burkitts disease, and the lymphoma cell line.
The IGF-IR, in addition to being implicated in nutritional support and in type-II diabetes, has also been associated with several types of cancers. For example, IGF-I has been implicated as an autocrine growth stimulator for several tumor types, e.g. human breast cancer carcinoma cells (Arteaga et al., 1989, J. Clin. Invest. 84:1418-1423) and small lung tumor cells (Macauley et al., 1990, Cancer Res. 50:2511-2517). In addition, IGF-I, integrally involved in the normal growth and differentiation of the nervous system, appears to be an autocrine stimulator of human gliomas. Sandberg-Nordqvist et al., 1993, Cancer Res. 53:2475-2478. The importance of the IGF-IR and its ligands in cell proliferation is further supported by the fact that many cell types in culture (fibroblasts, epithelial cells, smooth muscle cells, T-lymphocytes, myeloid cells, chondrocytes, osteoblasts, the stem cells of the bone marrow) are stimulated to grow by IGF-I. Goldring and Goldring, 1991, Eukaryotic Gene Expression 1:301-326. In a series of recent publications, Baserga even suggests that IGF-I-R plays a central role in the mechanisms of transformation and, as such, could be a preferred target for therapeutic interventions for a broad spectrum of human malignancies. Baserga, 1995, Cancer Res. 55:249-252; Baserga, 1994, Cell 79:927-930; Coppola et al., 1994, Mol. Cell. Biol. 14:4588-4595.
The association between abnormalities in RTKs and disease are not restricted to cancer, however. For example, RTKs have been associated with metabolic diseases like psoriasis, diabetes mellitus, wound healing, inflammation, and neurodegenerative diseases. These diseases include, but are not limited to hypertension, depression, generalized anxiety disorder, phobias, post-traumatic stress syndrome, avoidant personality disorder, sexual dysfunction, eating disorders, obesity, chemical dependencies, cluster headache, migraine, pain, Alzheimer""s disease, obsessive-compulsive disorder, panic disorder, memory disorders, Parkinson""s disease, endocrine disorders, vasospasm, cerebellar ataxia, and gastrointestinal tract disorders. For example, the EGF-R is indicated in corneal and dermal wound healing. Defects in the Insulin-R and the IGF-1R are indicated in type-II diabetes mellitus. A more complete correlation between specific RTKs and their therapeutic indications is set forth in Plowman et al., 1994, DNandP 7:334-339.
Not only receptor type tyrosine kinases, but also many cellular tyrosine kinases (CTKs) including are, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr, yrk (reviewed by Bolen et al., 1992, FASEB J. 6:3403-3409) are involved in the proliferative and metabolic signal transduction pathway and thus in indications of the present invention. For example, mutated src (v-src) has been demonstrated as an oncoprotein (pp60v-src) in chicken. Moreover, its cellular homolog, the proto-oncogene pp60c-src transmits oncogenic signals of many receptors. For example, overexpression of EGF-R or HER2/neu in tumors leads to the constitutive activation of pp60c-src, which is characteristic for the malignant cell but absent from the normal cell. On the other hand, mice deficient for the expression of c-src exhibit an osteopetrotic phenotype, indicating a key participation of c-src in osteoclast function and a possible involvement in related disorders. Similarly, Zap 70 is implicated in T-cell signaling.
Furthermore, the identification of CTK modulating compounds to augment or even synergize with RTK aimed blockers is an aspect of the present invention.
Finally, both RTKs and non-receptor type kinases have been connected to hyperimmune disorders.
II. The KDR/FLK-1 Receptor and VEGF
Normal vasculogenesis and angiogenesis play important roles in a variety of physiological processes such as embryonic development, wound healing, organ regeneration and female reproductive processes such as follicle development in the corpus luteum during ovulation and placental growth after pregnancy. Folkman and Shing, 1992, J. Biological Chem. 267:10931-34. However, many diseases are driven by persistent unregulated or inappropriate angiogenesis. For example, in arthritis, new capillary blood vessels invade the joint and destroy the cartilage. In diabetes, new capillaries in the retina invade the vitreous, bleed and cause blindness. Folkman, 1987, in: Congress of Thrombosis and Haemostasis (Verstraete, et. al, eds.), Leuven University Press, Leuven, pp.583-596. Ocular neovascularization is the most common cause of blindness and dominates approximately twenty (20) eye diseases.
Moreover, vasculogenesis and/or angiogenesis have been associated with the growth of malignant solid tumors and metastasis. A tumor must continuously stimulate the growth of new capillary blood vessels for the tumor itself to grow. Furthermore, the new blood vessels embedded in a tumor provide a gateway for tumor cells to enter the circulation and to metastasize to distant sites in the body. Folkman, 1990, J. Natl. Cancer Inst. 82:4-6; Klagsbrunn and Soker, 1993, Current Biology 3:699-702; Folkman, 1991, J. Natl., Cancer Inst. 82:4-6; Weidner et al., 1991, New Engl. J. Med. 324:1-5.
Several polypeptides with in vitro endothelial cell growth promoting activity have been identified. Examples include acidic and basic fibroblastic growth factor (aFGF, bFGF), vascular endothelial growth factor (VEGF) and placental growth factor. Unlike aFGF and bFGF, VEGF has recently been reported to be an endothelial cell specific mitogen. Ferrara and Henzel, 1989, Biochem. Biophys. Res. Comm. 161:851-858; Vaisman et al., 1990, J. Biol. Chem. 265:19461-19566.
Thus, the identification of the specific receptors to which VEGF binds is an important advancement in the understanding of the regulation of endothelial cell proliferation. Two structurally closely related RTKs have been identified to bind VEGF with high affinity: the flt-1 receptor (Shibuya et al., 1990, Oncogene 5:519-524; De Vries et al., 1992, Science 255:989-991) and the KDR/FLK-1 receptor, discussed in the U.S. patent application Ser. No. 08/193,829. Consequently, it had been surmised that these RTKs may have a role in the modulation and regulation of endothelial cell proliferation.
Evidence, such as the disclosure set forth in copending U.S. application Ser. No. 08/193,829, strongly suggests that VEGF is not only responsible for endothelial cell proliferation, but also is a prime regulator of normal and pathological angiogenesis. See generally, Klagsburn and Soker, 1993, Current Biology 3:699-702; Houck et al., 1992, J. Biol. Chem. 267:26031-26037. Moreover, it has been shown that KDR/FLK-1 and flt-1 are abundantly expressed in the proliferating endothelial cells of a growing tumor, but not in the surrounding quiescent endothelial cells. Plate et al., 1992, Nature 359:845-848; Shweiki et al., 1992, Nature 359:843-845.
II. Identification of Agonists and Antagonists to the KDR/FLK-1 Receptor
In view of the deduced importance of RTKs in the control, regulation and modulation of endothelial cell proliferation and potentially vasculogenesis and/or angiogenesis; many attempts have been made to identify RTK xe2x80x9cinhibitorsxe2x80x9d using a variety of approaches. These include the use of mutant ligands (U.S. Pat. No. 4,966,849); soluble receptors and antibodies (Application No. WO 94/10202; Kendall and Thomas, 1994, Proc. Natl. Acad. Sci. USA 90:10705-10709; Kim et al., 1993, Nature 362:841-844); and RNA ligands (Jellinek et al., 1994, Biochemistry 33:10450-10456).
Furthermore, tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO 91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani et al., 1994, Proc. Am. Assoc. Cancer Res. 35:2268), and inhibitors acting on receptor tyrosine kinase signal transduction pathways, such as protein kinase C inhibitors have been identified (Schuchter et al., 1991, Cancer Res. 51:682-687); Takano et al., 1993, Mol. Bio. Cell 4:358A; Kinsella et al., 1992, Exp. Cell Res. 199:56-62; Wright et al., 1992, J. Cellular Phys. 152:448-57).
More recently, attempts have been made to identify small molecules which act as tyrosine kinase inhibitors. For example, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642), vinylene-azaindole derivatives (PCT WO 94/14808) and 1-cyclopropyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992) have been described generally as tyrosine kinase inhibitors. Styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives (EP Application No. 0 566 266 A1), selenoindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO 91/15495) have been described as compounds for use as tyrosine kinase inhibitors for use in the treatment of cancer.
Consequently, there is an unmet need for the identification and generation of effective small compounds which selectively inhibit the signal transduction of the KDR/FLK-1 receptor in order to effectively and specifically suppress vasculogenesis.
Some of the compounds of the present invention demonstrate excellent activity in biological assays and thus these compounds and related compounds are expected to be effective in treating Flk related disorders such as those driven by persistent unregulated or inappropriate angiogenesis.
IV. Pharmaceutical Formulations and Routes of Administration
The compounds described herein can be administered to a human patient per se, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s). Techniques for formulation and administration of the compounds of the instant application may be found in xe2x80x9cRemington""s Pharmaceutical Sciences,xe2x80x9d Mack Publishing Co., Easton, Pa., latest edition.
a) Routes of Administration
Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a solid tumor, often in a depot or sustained release formulation.
Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tumor-specific antibody. The liposomes will be targeted to and taken up selectively by the tumor.
b) Composition/Formulation
The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks""s solution, Ringer""s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral admninistration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
A pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:D5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone, and other sugars or polysaccharides may substitute for dextrose.
Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
The pharmaceutical compositions also may comprise suitable solid or gel phase carries or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Many of the PTK modulating compounds of the invention may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
c) Effective Dosage
Pharmaceutical compositions suitable for use in the present invention include compositions where the active ingredients are contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the PTK activity). Such information can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient""s condition. (See e.g., Fingl et al., 1975, in xe2x80x9cThe Pharmacological Basis of Therapeuticsxe2x80x9d, Ch. 1 p.1).
Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the kinase modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data; e.g., the concentration necessary to achieve 50-90% inhibition of the kinase using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.
In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.
The amount of composition administered will, of course, be dependent on the subject being treated, on the subject""s weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
d) Packaging
The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the polynucleotide for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Suitable conditions indicated on the label may include treatment of a tumor, inhibition of angiogenesis, treatment of fibrosis, diabetes, and the like.
V. Biological Activity of the Indolinone Compounds
Indolinone compounds of the present invention were tested for their ability to inhibit most of protein tyrosine kinase activity. The biological assays and results of these inhibition studies are reported herein. The methods used to measure indolinone compound modulation of protein kinase function are similar to those described in U.S. application Ser. No. 08/702,232, by Tang et al., and entitled xe2x80x9cIndolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease,xe2x80x9d (Lyon and Lyon), filed Aug. 23, 1996, with respect to the high throughput aspect of the method. The Ser. No. 08/702,232 application is incorporated herein by reference in its entirety, including any drawings.
VI. Pharmaceutical Compositions and Administration of Indolinone Compounds
Methods of preparing pharmaceutical formulations of the compounds, methods of determining the amounts of compounds to be administered to a patient, and modes of administering compounds to an organism are disclosed in U.S. application Ser. No. 08/702,232, by Tang et al., and entitled xe2x80x9cIndolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease,xe2x80x9d (Lyon and Lyon), filed Aug. 23, 1996, and International patent publication number WO 96/22976, by Buzzetti et al., and entitled xe2x80x9cHydrosoluble 3-Arylidene-2-Oxindole Derivatives as Tyrosine Kinase Inhibitors,xe2x80x9d published Aug. 1, 1996, both of which are incorporated herein by reference in their entirety, including any drawings. Those skilled in the art will appreciate that such descriptions are applicable to the present invention and can be easily adapted to it.